This invention relates to the detection of DNA damage in eucaryotic organisms, and more specifically, to methods which quantitate the level of DNA chain breakage in individual cells.
DNA is a relatively fragile molecule which can be easily damaged in vivo by a number of different elements. For example, bending or shear forces can result in single-strand and double-strand breaks; a local pH change may cause the loss of constituent purine bases; chemical agents from the environment may modify one or more bases; and X-rays or ultraviolet (UV) radiation may bring about a chemical change in a base. Such changes in DNA may also be the result of radicals generated by normal metabolism. This kind of damage may be exacerbated by malnutrition, various toxins, or a subclinical disease state.
Nevertheless, DNA has the capacity to undergo faithful replication. The integrity of the information content of DNA is protected in large measure by enzymes capable of accurately repairing certain kinds of damage or defects in one strand of DNA so long as the other strand, which contains the complementary information, is intact.
Chromosomal DNA in mammalian cells is generally a continuous double helix over most of its length. For this reason it may be supercoiled Bases which are damaged, altered, or mismatched are subject to an intracellular repair mechanism called the excision-repair process, which involves the sequential action of several enzyme activities illustrated in FIG. 1 for the case of one special kind of damage. The defect is initially detected by a DNA polymerase which has a patrolling function, and which makes an incision on one side of the defect, thereby breaking one of the two polynucleotide chains which form the double helix. Following the removal of the damaged base(s), as well as many of the surrounding nucleotides, resynthesis of the DNA strand occurs using the undamaged, complementary chain as a template. The defective fragment is enzymatically excised, and the remaining nick is then religated to restore the continuity of the strand and the structure of the double helix. Thus, DNA damage of many types results in a transient strand-breakage which is part of the repair process. The repaired DNA is "as good as new" provided that the excision-repair process proceeds normally.
However, perfect repair does not always occur. Incorrect nucleotides are inserted at a low frequency, thereby resulting in somatic mutations that are perpetuated by each round of cellular division. The measurement of the rate of cellular DNA damage has assumed increasing importance because it appears that the accumulation of such damages, or resulting somatic mutations, can render the genome even more vulnerable to subsequent mutation, and may play a central role in degenerative diseases, cancer and aging.
Since different types of damage result in transient strand breakage, the incidence of strand breakage is an integrated measure of DNA damage. The speed of the cut, resynthesis, and religation processes limit the time over which the damage events may be registered as strand-breaks, but the repair process as a whole may be inhibited or may not exist at all in certain types of cells. Thus, the "not yet repaired window may be of variable width depending on cell type.
The importance of strand breakage, particularly as a consequence of radiation, has stimulated a great deal of work leading to measures of DNA damage. The procedures for damage assessment developed in the prior art include: alkaline sedimentation, alkaline unwinding (Storer et al. (1984) Anal. Biochem 142:351-359), filter elution (Kohn et al. (1976) Biochem. 15:4629-4637; Murray et al. (1987) Anal. Biochem. 160:149-159), and nucleoid sedimentation (Cuiffo et al. (1985) J. Free Radicals in Biol. Med. 1:139-144). However, these methods require about 10.sup.6 cells, and do not represent potential methods for determining low level radical damage in intact animals or humans.
An estimation of DNA damage has been calculated from a determination of the amounts of oxidized bases and nucleotides excised from the DNA by the excision-repair process and subsequently collected in the urine (Carthcart et al. (1984) Proc. Natl. Acad. Sci. USA 81:5633-5637; Adelman et al. (1988) Proc. Natl. Acad. Sci. USA 85:2706-2708). Using this method, it has been determined that most cells are subject to a surprisingly high rate of damage. Moreover, this rate of damage is proportionately higher in mammals having higher metabolic rates.
However, from an experimental and diagnostic point of view, it would be highly desirable to be able to measure the damage that an individual cell of identifiable type has sustained, since the rate of damage is likely to be variable depending on the individual donor, cell type, and cell age as well as state of activation. Earlier work on single cells has been based upon the lysing cells embedded in agarose under alkaline conditions, (Rydberg et al. (1978) DNA Repair Mechanisms, Hanawold and Friedberg, eds. pp. 465-468), followed by electrophoresis under neutral or alkaline conditions (Ostling et al. (1984) Biochem. Biophys. Res. Commun. 123:291-298; Singh et al. (1988) Expt. Cell Res. 175:184-191). The extent of DNA damage is determined by comparing the migration pattern of the DNA with that of control DNA. These methods are sensitive, but have the drawback that the embedding procedure requires time, loses track of individual cells, denatures the DNA, and changes the conditions of their surface contacts. Pardoll et al. (Cell (1980) 19:527-536) and Cook et al. (J. Cell Sci. (1976 ) 22:303-324) describe the formation of a nuclear matrix structure composed mainly of undenatured DNA, but neither suggest being able to determine the presence or extent of DNA damage from such a structure.
Therefore, it is an object of the invention to provide a simple and efficient method of determining the extent and rate of DNA damage occurring in an animal or human.
It is also an object of the invention to provide a simple procedure to measure the incidence of DNA chain breakage in an individual cell.
It is another object of the invention to provide a sensitive method for detection of DNA chain breakage which can be applied before the natural repair and religation process is completed.
These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.